1. Field of the Invention
The present invention relates to a method for purifying a defined amount of nucleic acids from a sample containing nucleic acids. Furthermore, suitable kits and nucleic acid binding phases are provided, which allow a defined amount of nucleic acids to be isolated from a sample and are therefore suitable for normalizing the nucleic acid concentration in the eluate.
2. Description of Related Art
Various methods for purifying and isolating nucleic acids are known in the prior art. These include the use of phenol-chloroform, salting-out techniques, the use of ion exchangers and silica particles. A known method of nucleic acid purification is the so-called “charge-switch method”. According to this, a nucleic acid binding phase at a first pH is brought into contact with a nucleic acid-containing sample, wherein the nucleic acid binding phase has a positive charge. This promotes binding of the negatively charged nucleic acids to the phase. For release/elution of the nucleic acids, according to the charge-switch principle a second pH is established, which is higher than the pKs value of the nucleic acid binding phase, in order to invert, i.e. neutralize, the positive charge. This promotes detachment of the bound nucleic acids from the nucleic acid binding phase.
For many analytical techniques and biological methods it is necessary to use a certain, i.e. defined amount of nucleic acid. The amount of nucleic acid isolated or amplified from different starting materials (samples) depends on a number of factors that are difficult to control, so that yields can vary widely depending on method and material. For subsequent experiments with the isolated nucleic acids to be carried out under comparable conditions, as a rule this requires quantification of the nucleic acid obtained and then adjustment of the concentrations or amount of nucleic acid to a specified value and therefore normalization. For many standardized processes and in particular process automation, such normalization is often essential, in order to obtain reproducible results.
For this reason there are a large number of methods for quantifying nucleic acids in a purified sample. One example of a usual quantification method is the spectrophotometric determination of the amount of DNA in a sample. Following determination of the concentration of the nucleic acid in the sample, the concentration can then be adjusted uniformly. Other known methods for quantifying nucleic acids are based on intercalating dyes, e.g. ethidium bromide, SYBR Green or Picogreen. The concentration can be determined by comparing the measured values with a standard curve.
In addition to the spectrometric or fluorescence-based quantification techniques described, various purification techniques are also known in the prior art, which are said to allow uniform concentrations of nucleic acids to be prepared from different starting materials. Examples are the MagneSil-Kit from Promega or the SequalPrep-Kit from Invitrogen.
The MagneSil-Kit from Promega is used, for example, for isolating genomic DNA from whole blood. The kit is based on the binding of DNA to silica-coated paramagnetic particles under chaotropic salt conditions. As a result, usually 1 μg (±50%) of purified genomic DNA is obtained after elution with a low-salt buffer or water. Due to the relatively large fluctuations of up to 50%, this method requires improvement.
The SequalPrep-Kit from Invitrogen is based on binding of DNA to surfaces that are coated with an ion exchanger. The DNA binds at an acid pH and elution is carried out with a strongly alkaline buffer, the pH of which is above the pKs value of the ion exchanger. This kit is based on the so-called charge-switch technology (see above). The kit finds application in the purification and normalization of long range (LR) PCRs and it is also suitable for template preparation for sequencings. The manufacturer states yields of approx. 25 ng with a two to three-fold fluctuation when using at least 250 ng starting DNA. This means that also here the fluctuations can also be relatively large, so that once again optimization is required.
The known purification techniques therefore in practice still have wide variations in yields. Difficulties arise in particular when different amounts of starting material or different starting materials are used.